Research Article: A Statistical Evaluation of Methods of In-Vitro Growth Assessment for Phyllosticta citricarpa: Average Colony Diameter vs. Area

Date Published: January 26, 2017

Publisher: Public Library of Science

Author(s): Katherine E. Hendricks, Mary C. Christman, Pamela D. Roberts, Sabrina Sarrocco.


Fungal growth inhibition on solid media has been historically measured and calculated based on the average of perpendicular diameter measurements of growth on fungicide amended media. We investigated the sensitivity of the calculated area (DA) and the measured area (MA) for assessing fungicide growth inhibition of the ascomycete, Phyllosticta citricarpa on solid media. Both the calculated, DA and the actual measured area, MA were adequate for distinguishing significant treatment effects of fungicide on fungal growth, however MA was more sensitive at identifying significant differences between the controls and fungicide concentrations below 5 ppm.

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Methods of filamentous fungal growth assessment depend upon the media in which the fungus is grown. Direct measurements of fungal growth are utilized when fungi are grown in liquid media and single time point measurements are required. Biomass production (fungal growth) can be measured directly from dry or wet weight assessment. In cases where there is difficulty in separating mycelia from media, indirect measures are utilized. Indirect measures of fungal growth include visual observation and measurement [1–6], indicator dye hydrolysis, increases in measurable component unique to the fungus such as growth linked enzymes—laccase, ergosterol and/or chitin [7–9] and absorbance [10–13]. Radial growth measurement is a popular indirect measure of filamentous growth on solid media [14–16] when assessing fungicidal properties of chemicals. Radial growth measurements are usually taken perpendicularly [17] and may be reported as the average of two or more pairs of measurements [6]. Measurements may be made utilizing pre-drawn lines prior to placement of the inoculating fungal plug or drawn at the time of assessment.

In comparing methods of assessment of fungal growth on control media and media amended with fenbuconazole using inhibition ratios, the fixed effect Media*Day*Method was highly significant for Ratio0 and RatioA, having p-values of p<0.0001 (for Ratio0, F23, 2033 = 259.27; for RatioA, F23, 2033 = 273.35). Measurements of fungal growth inhibition by measured area (MA) were significantly different from calculated area, DA at lower concentrations of fungicide (0.1 to 5 ppm) on all days measured with the exception of 5 ppm on Day 7 (Fig 2A). This can be attributed to the initial lag or inhibition in growth caused by higher concentration of the fungicide. There was no difference between methods used to assess fungicide growth inhibition for the 10 ppm on all days assessed (Fig 3; S1 Table) and this is attributed to complete inhibition of growth by the highest fungicide concentration. This was true for ratios calculated based on either PDA (Ratio0) or PDA amended with acetone (RatioA) as controls (Fig 2A and 2B).   Source: